The present invention relates to DNA chains which are useful for synthesis of carotenoids suitably used for reviving the colors of farm-raised fish such as sea bream, shrimp and salmon, and hen eggs, and for synthesis of carotenoids such as astaxanthin which is applicable as a coloring agent or antioxidant to foods, and to methods of producing carotenoids such as astaxanthin utilizing microorganisms incorporating such DNA chains.
In the natural word, over 600 of different carotenoids have been identified from plants, microorganisms and the like. Industrially useful carotenoids are generally produced by chemical synthesis processes for which possibility of undesired actions such as contamination of synthesis auxiliary materials is feared. In addition, tastes of consumers tend to lean toward naturally-occurring carotenoids. However, there is a limit to extraction from plants and the like natural products, and an effective industrial process is not entirely established. As a production method of naturally-occurring carotenoids, microbial fermentation methods have been reported in some cases, however, none of such cases enable production of carotenoids in an amount which is enough for economical industrial production. Likewise the cases of carotenoids, when trying to produce a functional substance from a microorganism, one will choose a microorganism which serves as a host of fermentation by broad screening. Then, in many cases, through classical mutation and breeding using a chemical treatment agent, a highly productive strain is isolated and subjected to production or research, because a production amount from a wild-type of carotenoid producing microorganism is usually small.
As a microorganism that produces useful carotenoid, Yokoyama et al. reported Agrobacterium (later, reclassified into bacteria belonging to Paracoccus) marine bacteria (Non-patent document 1). These strains are characterized by synthesizing astaxanthin which is a functional carotenoid in high content. As described above, a production amount of astaxanthin or the like of Paracoccus bacterial can be increased through mutation process, and a strain TSN18E7 with improved production amount (see Japanese Patent Laid-Open Publication 2005-58216) is deposited to International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the number of FERM P-19746.
A carotenoid biosynthesis pathway is made up of various enzymes, and genes encoding such enzymes have been analyzed by many researches. In a typical pathway, for example, carotenoid is synthesized in its early stage by an isoprenoid biosynthesis pathway which is shared by steroid and terpenoid, starting from mevalonic acid which is a basic metabolite. Farnesyl pyrophosphate having 15 carbons (C15) generating through the isoprenoid basic synthesis system is condensed with isopentenyl diphosphate (IPP) (C5), to give geranylgeranyl diphosphate (GGPP) (C20). Then through condensation of two molecules of GGPP, colorless phytoene which is the first carotenoid is synthesized. The phytoene is then converted into lycopene through a series of unsaturation reactions, and then the lycopene is converted into β-carotene through a cyclization reaction. Then, a hydroxyl group and a keto group are introduced into the β-carotene, which leads synthesis of various xanthophylls represented by astaxanthin (FIG. 1).
From these gene level findings, studies intended to improvement of carotenoid synthesis with the use of genetic recombination technique have been made. See Chia-wei Wang et al., Biotechnol. Prog., 16: 922-926 (2000); Claudia Schmidt-Dannert et al., Nat. Biotechnol., 18: 750-753 (2000); Daisuke Umeno et al., Appl. Environ. Microbiol., 69: 3573-3579 (2003), for example. In these studies, Escherichia coli that does not synthesize carotenoid is used as a host, so that it would be difficult to apply these studies to industrial production because of their low productivity of carotenoid. In other report, increase in carotenoid synthesis amount is realized by introducing a carotenoid gene into a bacterium that produces carotenoid (Patent document 1). However, it would be still difficult to apply such prepared gene recombinant strain to industrial production because of its low amount of carotenoid synthesis.    [Non-patent document 1] Yokoyama, A. H. Izumida, and W. Miki, Production of astaxanthin and 4-ketozeaxanthin by the marine bacterium, Agrobacterium aurantiacum, Biosci. Biotechnol. Biochem., 58: 1842-1844 (1994).    [Non-patent document 2] Norihiko Misawa, Yoshiko Satomi, Keiji Kondo, Akihiro Yokoyama, Susumu Kajiwara, Tochiko Saito, Takeshi Ohtani, and Wataru Miki, Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level, J., Bacteriology 177: 6575-6584 (1995).    [Non-patent document 3] Eric A. Johnson, and William A. Schroeder, Microbial Carotenoids, Advances in Biochemical Engineering Biotechnology, 53: 119-178 (1995).    [Non-patent document 4] P. C. Lee, and Schmidt-Dannert, Metabolic engineering towards biotechnological production of carotenoids in microorganism, 60:1-11 (2002).    [Non-patent document 5] Kovach, M. E. et al., GENE166, 175-176 (1995).    [Non-patent document 6] R. Simon, U. Priefer, and A. Puhler, A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria, BIO/TECHNOLOGY, 1: 784-791 (1983).    [Non-patent document 7] Cedric Y. Szpiper, Michel Faelen, and Martine Couturier, Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1, J. Bacteriology, 183: 2101-2110 (2001).    [Patent document 1] Japanese translation of PCT application JP-A 2004-527265    [Patent document 2] Japanese Patent Publication No. 3403381    [Patent document 3] Japanese Patent Application No. 2005-106045